The ability to stably transduce lymphohematopoietic stem-progenitor cells (HSCs) and progeny cells allows us to genetically engineer HSCs and progeny, especially dendritic cells (DCs), to serve as cellular tools to improve transplantation. Allogeneic (allo) blood and marrow transplantation (BMT) will continue to be an important treatment option in patients with many inherited and acquired diseases. Our recent Preliminary Results indicate that ex vivo or in vivo treatment with transduced FasL+ allog mouse DCs or HSCs suppressed the response to that alloantigen and increased engraftment of allo cells, without immunoablation or evident toxicity to the recipient mice. In Aims 1-2 of this proposal, we propose studies to test the concept and elucidate the principal mechanisms by which transduced DCs or HSCs constitutively expressing Fas ligand (FasL) appear to reduce the T (and NK) cells that mediate graft rejection without overwhelming organ toxicity or general immunologic impairment. Aims 1-2 attempt to accelerate and enhance the natural homeostatic role of FasL during the course of the alloimmune response, with the potential outcome that alloimmune T cells may be killed earlier in their activation/expansion, or killed more potently later during activation-induced cell death (AICD), by transduced FasL+ DCs (Aim 1) or HSCs/progeny (Aim 2), respectively. As we investigate this, we will examine the apoptotic pathways in alloimmune cells. In addition, since we expect that transduced FasL+ HSCs might potentially be toxic in vivo, we will in parallel investigate technologies to limit potential FasL toxicity, eg by transducing only a small percent of high quality HSCs, by employing a FasL deletion mutant that cannot be cleaved to release soluble FasL (sFasL), by eliminating the transduced cells themselves or their FasL expression after tolerance to HSCs has been generated, or by using lineage/stage-specific promoters to restrict FasL expression. A novel FasL+ cell therapy approach to reduce graft rejection may eventually be used in clinical allo BMT. Furthermore, achievement of stable lymphohematopoietic chimerism would also be predicted to generate tolerance for transplanted allo organs or pluripotent stem cells. Therefore, in Aim 3, we will assess tolerance to cardiac allografts in hematopoietic macro-chimeras, generated by this FasL strategy, with or without other immunosuppressive methodologies such as co-stimulatory blockade. SPECIFIC AIM 1: Assess whether FasL+ DCs specifically reduce an alloimmune response and enhance engraftment of allo HSCs SPECIFIC AIM 2: Determine whether FasL+ HSCs generate specific tolerance in allo BMT. SPECIFIC AIM 3: Evaluate tolerance to cardiac allografts in hematopoietic chimeras generated using FasL+ DCs/HSCs.